In mitochondria, substrate oxidation by multisubunit complexes of the electron transport chain is coupled to ATP synthesis by the ATP synthase complex via an electrochemical proton gradient, Delta-mu-H+, across the mitochondrial inner membrane. The ability to synthesize ATP, establish Delta-mu-H+ and catalyze the energy-linked reactions is lost in bovine heart submitochondrial particles sonicated at pH 8.8 in the presence of 0.6 mM EDTA, due to proton leakiness of the mitochondrial inner membrane. Recently, we cloned and expressed in bacteria a human mitochondrial polypeptide, factor B, that efficiently restored all the energy-linked reactions, including the ATP synthesis, in the pH 8.8/EDTA treated submitochondrial particles. Because factor B coupling activity is similar to that of low amounts of oligomycin, a specific inhibitor of proton translocation through the membrane sector F-0 of ATP synthase, we propose that factor B is a regulatory component of mitochondrial ATP synthase, loosely associated with F-0 sector. We hypothesize that in mitochondria, factor B is involved in regulation of proton flux through F-0 sector coupled to ATP synthesis in the active site of the enzyme. We further hypothesize that via redox status of its essential thiols, specifically a vicinal dithiol, factor B acts as a "pressure valve" for maintaining proton-motive force below the threshold that favors production of reactive oxygen species in mitochondria, by inducing a mild uncoupling of the organelle. The following specific aims are proposed: 1) To characterize the interaction of factor B with the membrane sector F-0 in the membrane bound and detergent solubilized ATP synthase complex; and 2) To elucidate the structure-function relationship of the vicinal dithiol motif found in the amino acid sequence of the mature factor B, and the role it plays in the factor B sensitivity toward Cd 2+ and phenylarsine oxide, both of which uncouple oxidative phosphorylation in mitochondria. The proposed research is expected to advance the knowledge of the molecular mechanisms of energy transfer and conservation in mitochondria, and shed light on the role of mitochondria in a number of human disorders.